Role of stress fiber-like structures in assembling nascent myofibrils in myosheets recovering from exposure to ethyl methanesulfonate

When day 1 cultures of chick myogenic cells were exposed to the mutagenic alkylating agent ethyl methanesulfonate (EMS) for 3 d, 80% of the replicating cells were killed, but postmitotic myoblasts survived. The myoblasts fused to form unusual multinucleated "myosheets": extraordinarily wide, flattened structures that were devoid of myofibrils but displayed extensive, submembranous stress fiber-like structures (SFLS). Immunoblots of the myosheets indicated that the carcinogen blocked the synthesis and accumulation of the myofibrillar myosin isoforms but not that of the cytoplasmic myosin isoform. When removed from EMS, widely spaced nascent myofibrils gradually emerged in the myosheets after 3 d. Striking co-localization of fluorescent reagents that stained SFLS and those that specifically stained myofibrils was observed for the next 2 d. By both immunofluorescence and electron microscopy, individual nascent myofibrils appeared to be part of, or juxtaposed to, preexisting individual SFLS. By day 6, all SFLS had disappeared, and the definitive myofibrils were displaced from their submembranous site into the interior of the myosheet. Immunoblots from recovering myosheets demonstrated a temporal correlation between the appearance of the myofibrillar myosin isoforms and the assembly of thick filaments. The assembly of definitive myofibrils did not appear to involve desmin intermediate filaments, but a striking aggregation of sarcoplasmic reticulum elements was seen at the level of each I-Z-band. Our findings suggest that SFLS in the EMS myosheets function as early, transitory assembly sites for nascent myofibrils.     

Scaffolds and chaperones in myofibril assembly: putting the striations in striated muscle

Sarcomere assembly in striated muscles has long been described as a series of steps leading to assembly of individual proteins into thick filaments, thin filaments and Z-lines. Decades of previous work focused on the order in which various structural proteins adopted the striated organization typical of mature myofibrils. These studies led to the view that actin and α-actinin assemble into premyofibril structures separately from myosin filaments, and that these structures are then assembled into myofibrils with centered myosin filaments and actin filaments anchored at the Z-lines. More recent studies have shown that particular scaffolding proteins and chaperone proteins are required for individual steps in assembly. Here, we review the evidence that N-RAP, a LIM domain and nebulin repeat protein, scaffolds assembly of actin and α-actinin into I-Z-I structures in the first steps of assembly; that the heat shock chaperone proteins Hsp90 & Hsc70 cooperate with UNC-45 to direct the folding of muscle myosin and its assembly into thick filaments; and that the kelch repeat protein Krp1 promotes lateral fusion of premyofibril structures to form mature striated myofibrils. The evidence shows that myofibril assembly is a complex process that requires the action of particular catalysts and scaffolds at individual steps. The scaffolds and chaperones required for assembly are potential regulators of myofibrillogenesis, and abnormal function of these proteins caused by mutation or pathological processes could in principle contribute to diseases of cardiac and skeletal muscles.     

Involvement of transglutaminase in myofibril assembly of chick embryonic myoblasts in culture

Involvement of transglutaminase in myofibrillogenesis of chick embryonic myoblasts has been investigated in vitro. Both the activity and protein level of transglutaminase initially decreased to a minimal level at the time of burst of myoblast fusion but gradually increased thereafter. The localization of transglutaminase underwent a dramatic change from the whole cytoplasm in a diffuse pattern to the cross- striated sarcomeric A band, being strictly colocalized with the myosin thick filaments. For a brief period prior to the appearance of cross- striation, transglutaminase was localized in nonstriated filamental structures that coincided with the stress fiber-like structures. When 12-o-tetradecanoyl phorbol acetate was added to muscle cell cultures to induce the sequential disassembly of thin and thick filaments, transglutaminase was strictly colocalized with the myosin thick filaments even in the myosacs, of which most of the thin filaments were disrupted. Moreover, monodansylcadaverine, a competitive inhibitor of transglutaminase, reversibly inhibited the myofibril maturation. In addition, myosin heavy chain behaved as one of the potential intracellular substrates for transglutaminase. The cross-linked myosin complex constituted approximately 5% of the total Triton X-100- insoluble pool of myosin molecules in developing muscle cells, and its level was reduced to below 1% upon treatment with monodansylcadaverine. These results suggest that transglutaminase plays a crucial role in myofibrillogenesis of developing chick skeletal muscle.     

Genetic analysis of myosin assembly inCaenorhabditis elegans

The established observations and unresolved questions in the assembly of myosin are outlined in this article. Much of the background information has been obtained in classical experiments using the myosin and thick filaments from vertebrate skeletal muscle. Current research is concerned with problems of myosin assembly and structure in smooth muscle, a broad spectrum of invertebrate muscles, and eukaryotic cells in general. Many of the general questions concerning myosin assembly have been addressed by a combination of genetic, molecular, and structural approaches in the nematode Caenorhabditis elegans. Detailed analysis of multiple myosin isoforms has been a prominent aspect of the nematode work. The molecular cloning and determination of the complete sequences of the genes encoding the four isoforms of myosin heavy chain and of the myosin-associated protein paramyosin have been a major landmark. The sequences have permitted a theoretical analysis of myosin rod structure and the interactions of myosin in thick filaments. The development of specific monoclonal antibodies to the individual myosins has led to the delineation of the different locations of the myosins and to their special roles in thick filament structure and assembly. In nematode body-wall muscles, two isoforms, myosins A and B, are located in different regions of each thick filament. Myosin A is located in the central biopolar zones, whereas myosin B is restricted to the flanking polar regions. This specific localization directly implies differential behavior of the two myosins during assembly. Genetic and structural experiments demonstrate that paramyosin and the levels of expression of the two forms are required for the differential assembly. Additional genetic experiments indicate that several other gene products are involved in the assembly of myosin. Structural studies of mutants have uncovered two new structures. A core structure separate from myosin and paramyosin appears to be an integral part of thick filaments. Multifilament assemblages exhibit multiple nascent thick filament-like structures extending from central paramyosin regions. Dominant mutants of myosin that disrupt thick filament assembly are located in the ATP and actin binding sites of the heavy chain. A model for a cycle of reactions in the assembly of myosin into thick filaments is presented. Specific reactions of the two myosin isoforms, paramyosin, and core proteins with multifilament assemblages as possible intermediates in assembly are proposed.     

In situ reconstitution of myosin filaments within the myosin-extracted myofibril in cultured skeletal muscle cells

We studied the in situ reconstitution of myosin filaments within the myosin-extracted myofibrils in cultured chick embryo skeletal muscle cells using the electron microscope and polarization microscope. Myosin was first extracted from the myofibrils in glycerinated muscle cells with a high-salt solution containing 0.6 M KCl. When rabbit skeletal muscle myosin was added to the myosin-extracted cells in the high-salt solution, thin filaments in the ghost myofibrils were bound with myosin to form arrowhead complexes. Subsequent dilution of KCl in the myosin solution to 0.1 M resulted in the formation of thick myosin filaments within the myofibrils, increasing the birefringence of the myofibrils. When Mg-ATP was added such myosin-reassembled myofibrils were induced either to form supercontraction bands or to restore the sarcomeric arrangement of thick and thin filaments. Under the polarization microscope, vibrational movement of the myofibrils was seen transiently upon addition of Mg-ATP, often resulting in a regular arrangement of myofibrils in register. These myofibrils, with reconstituted myosin filaments, structurally and functionally resembled the native myofibrils. The findings are discussed with special reference to the myofibril formation in developing muscle cells.     

Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres

Several biologically important protein structures give rise to strong second-harmonic generation (SHG) in their native context. In addition to high-contrast optical sections of cells and tissues, SHG imaging can provide detailed structural information based on the physical constraints of the optical effect. In this study we characterize, by biochemical and optical analysis, the critical structures underlying SHG from the complex muscle sarcomere. SHG emission arises from domains of the sarcomere containing thick filaments, even within nascent sarcomeres of differentiating myocytes. SHG from isolated myofibrils is abolished by extraction of myosin, but is unaffected by removal or addition of actin filaments. Furthermore, the polarization dependence of sarcomeric SHG is not affected by either the proportion of myosin head domains or the orientation of myosin heads. By fitting SHG polarization anisotropy readings to theoretical response curves, we find an orientation for the elemental harmonophore that corresponds well to the pitch of the myosin rod alpha-helix along the thick filament axis. Taken together, these data indicate that myosin rod domains are the key structures giving SHG from striated muscle. This study should guide the interpretation of SHG contrast in images of cardiac and skeletal muscle tissue for a variety of biomedical applications.     

FINE STRUCTURE AND SIZE DISTRIBUTION OF FREE THICK FILAMENTS IN EARLY FIBRILLOGENESIS OF ASCIDIAN TADPOLE

The development and the size distribution of free thick filaments which accumulate in the early stages of myofibril formation in somitic myoblasts of the ascidian tadpole were studied by electron microscopy. Such filaments appeared in the cell cortex but, rather dominantly, the aggregates of these thick filaments and filamentous structures were observed in the interior of the cell. The aggregate consisted of some of the following elements: filamentous structures (20–60 A in diameter); free thick filaments (60–220 A); dense Z-band precursor materials; bundles of thick (140–160 A) and thin (60–70 A) filaments; and ribosomal clusters. The free thick filaments were variable in diameter and showed long lateral projections (300–600 A) and tapered ends.
The variation curve in diameter of the free thick filaments indicates a continuous size distribution, suggesting the continuous growth of these filaments by polymerization of myosin molecules. Free thick filaments thicker than myosin filaments which were found within myofibrils were present; their significance is discussed in relation to myosin filament formation.     

Incorporation of fluorescently labeled contractile proteins into freshly isolated living adult cardiac myocytes.

When fluorescently labeled contractile proteins are injected into embryonic muscle cells, they become incorporated into the cells' myofibrils. In order to determine if this exchange of proteins is unique to the embryonic stage of development, we isolated adult cardiac myocytes and microinjected them with fluorescently labeled actin, myosin light chains, alpha-actinin, and vinculin. Each of these proteins was incorporated into the adult cardiomyocytes and was colocalized with the cells' native proteins, despite the fact that the labeled proteins were prepared from noncardiac tissues. Within 10 min of injection, alpha-actinin was incorporated into Z-bands surrounding the site of injection. Similarly, 30 sec after injection, actin was incorporated into the entire I-bands at the site of injection. Following a 3-h incubation, increased actin fluorescence was noted at the intercalated disc. Vinculin exchange was seen in the intercalated discs, as well as in the Z-bands throughout the cells. Myosin light chains required 4-6 h after injection to become incorporated into the A-bands of the adult muscle. Nonspecific proteins, such as fluorescent BSA, showed no association with the myofibrils or the former intercalated discs. When adult cells were maintained in culture for 10 days, they retain the ability to incorporate these contractile proteins into their myofibrils. T-tubules and the sarcoplasmic reticulum could be detected in periodic arrays in the freshly isolated cells using the membrane dye WW781 and DiOC6[3], respectively. In conclusion, the myofibrils in adult, as in embryonic, muscle cells are dynamic structures, permitting isoform transitions without dismantling of the myofibrils.     

Posttranslational incorporation of contractile proteins into myofibrils in a cell-free system

The incorporation of newly synthesized protein into myofibrils has been examined in a cell-free system. Myofibrils were added to a reticulocyte lysate after the in vitro translation of muscle-specific poly(A)+RNA. Only a small number of the many synthesized proteins were found to associate with the exogenously added myofibrils. These proteins were all identified as sarcomeric components and had subunit mobilities (Mr) of 200, 140, 95, 86, 43, 38, 35, 25, 23, 20, and 18 kD. The association was rapid (t1/2 less than 15 min) and, for most of the proteins, relatively temperature insensitive. Except for a 43-kD polypeptide, tentatively identified as beta-actin, none of the proteins encoded by brain poly(A)+RNA associated with the myofibrils. When filaments made from purified myosin or actin were used as the "capture" substrates, only thick or thin filament proteins, respectively, were incorporated. Incorporation was substantially reduced when cross-linked myosin filaments were used. These results are compatible with a model in which proteins of the sarcomere are in kinetic equilibrium with homologous proteins in a soluble pool.     

Differential response of myofibrillar and cytoskeletal proteins in cells treated with phorbol myristate acetate

Muscle-specific and nonmuscle contractile protein isoforms responded in opposite ways to 12-o-tetradecanoyl phorbol-13-acetate (TPA). Loss of Z band density was observed in day-4-5 cultured chick myotubes after 2 h in the phorbol ester, TPA. By 5-10 h, most I-Z-I complexes were selectively deleted from the myofibril, although the A bands remained intact and longitudinally aligned. The deletion of I-Z-I complexes was inversely related to the appearance of numerous cortical, alpha-actinin containing bodies (CABs), transitory structures approximately 3.0 microns in diameter. Each CAB consisted of a filamentous core that costained with antibodies to alpha-actin and sarcomeric alpha-actinin. In turn each CAB was encaged by a discontinuous rim that costained with antibodies to vinculin and talin. Vimentin and desmin intermediate filaments and most cell organelles were excluded from the membrane-free CABs. These curious bodies disappeared over the next 10 h so that in 30-h myosacs all alpha-actin and sarcomeric alpha-actinin structures had been eliminated. On the other hand vinculin and talin adhesion plaques remained prominent even in 72-h myosacs. Disruption of the A bands was first initiated after 15-20 h in TPA (e.g., 15-20-h myosacs). Thick filaments of apparently normal length and structure were progressively released from A segments, and by 40 h all A bands had been broken down into enormous numbers of randomly dispersed, but still intact single thick filaments. This breakdown correlated with the formation of amorphous cytoplasmic aggregates which invariably colocalized antibodies to myosin heavy chain, MLC 1-3, myomesin, and C protein. Complete elimination of all immunoreactive thick filament proteins required 60-72 h of TPA exposure. The elimination of the thick filament-associated proteins did not involve the participation of vinculin or talin. In contrast to its effects on myofibrils, TPA did not induce the disassembly of the contractile proteins in stress fibers and microfilaments either in myosacs or in fibroblastic cells. Similarly, TPA, which rapidly induces the translocation of vinculin and talin to ectopic sites in many types of immortalized cells, had no gross effect on the adhesion plaques of myosacs, primary fibroblastic cells, or presumptive myoblasts. Clearly, the response to TPA of contractile protein and some cytoskeletal isoforms not only varies among phenotypes, but even within the domains of a given myotube the myofibrils respond one way, the stress fibers/microfilaments another.     

Identification and localization of high molecular weight proteins in insect flight and leg muscle.

Thick and thin filaments in asynchronous flight muscle overlap nearly completely and thick filaments are attached to the Z-disc by connecting filaments. We have raised antibodies against a fraction of Lethocerus flight muscle myofibrils containing Z-discs and associated filaments and also against a low ionic strength extract of myofibrils. Monoclonal antibodies were obtained to proteins of 800 kd (p800), 700 kd (p700), 400 kd (p400) and alpha-actinin. The positions of the proteins in Lethocerus flight and leg myofibrils were determined by immunofluorescence and electron microscopy. p800 is in connecting filaments of flight myofibrils and in A-bands of leg myofibrils. p700 is in Z-discs of flight myofibrils and an immunologically related protein, p500, is in leg muscle Z-discs. p400 is in M-lines of both flight and leg myofibrils. Preliminary DNA sequencing shows that p800 is related to vertebrate titin and nematode twitchin. Molecules of p800 could extend from the Z-disc a short way along thick filaments, forming a mechanical link between the two structures. All three high molecular weight proteins probably stabilize the structure of the myofibril.     

Modulation of myosin assembly

Myosin self-assembly is generally considered to be the major process in thick filament formation within striated muscles. The biological assembly of myosin into thick filaments is being analysed by genetic dissection as well as biochemical and morphological experiments in the nematode Caenorhabditis elegans. This work shows that the assembly of myosin is modulated by its biosynthesis and interaction with non-myosin proteins. Assemblages which generate multiple nascent thick filaments may play a central role in a catalytic cycle of myosin assembly.     

Developmental relationship of myosin binding proteins (myomesin, connectin and C-protein) to myosin in chicken somites as studied by immunofluorescence microscopy

The developmental relationship of myosin binding proteins (myomesin, connectin and C-protein) to myosin was studied in chicken cervical somites by immunofluorescence microscopy. Muscle and non-muscle myosins initially appeared as slender rods at the same sites, and then, fused to form non-striated fibrils. As muscle myosin formed striated structures (A bands), non-muscle myosin disappeared from this structure. Myomesin (reactive with monoclonal antibodies MyB4 and MyBB78) and connectin (carboxy terminal region, reactive with monoclonal antibody T51) were seen as dots in the center of these myosin rods. These proteins then formed characteristic mature striations on non-striated fibrils of myosin. Earlier alignment of these myosin binding proteins rather than myosin indicates that the correct assembly of these proteins seems to be related to the formation of initial myosin rods as well as subsequent linear and periodic alignment of myosin molecules to form early A bands. Connectin spots reactive with 9D10 were scattered around myosin rods/myomesin dots/connectin T51 dots. These spots may represent radiating connectin filaments from these rods/dots to link myosin rods to the I-Z-I structures of myofibrils to be incorporated. Since the slow isoform of C-protein formed its characteristic bands ("doublets") prior to H zone formation within A bands by myosin, this isoform may help to precisely align myosin filaments within the A band region. The presence of the slow, then the slow and the cardiac, and finally the co-existence of the slow and the fast isoforms of C-protein may interfere with the incorporation and co-polymerization of non-adult isoforms into myofibrils.     

Cytoskeletal filaments in embryonic chick myocardial cells as revealed by the quick-freeze deep-etch method combined with immunocytochemistry

Summary The three-dimensional organization of cytoskeletal filaments associated with the myofibrils and sarcolemma of the myocardial cells of early chick embryos was studied by the rapid-freeze deep-etch method combined with immunocytochemistry. In the endoplasmic region of saponin-treated myocardial cells, 12–14 nm filaments formed a loose network surrounding nascent myofibrils. These 12–14 nm filaments attached to the myofibrils and some of them converged into Z disc regions. In the non-junctional cytocortical region thinner 8–11 nm filaments composed a dense network just beneath the sarcolemma. In myofibril terminating regions at the sarcolemma, i.e., the fascia adherens, 3–5 nm cross-bridges were observed among the thin filaments. In Triton-permeabilized and myosin subfragment 1 (S1)-treated samples, subsarcolemmal 8–11 nm filaments proved to be S1-decorated actin filaments under which there was a loose network of S1-undecorated filaments. Subsarcolemmal S1-decorated actin filaments had mixed polarity and attached to the sarcolemma at one end. A loose network of S1-undecorated filaments among myofibrils in the endoplasmic region was revealed to consist of desmin-containing intermediate filaments after immuno-gold staining for desmin. These networks connecting myofibrils with sarcolemma were assumed to play an important role in integrating and transmitting the contractile force of individual myofibrils within early embryonic myocardial cells.     

Contribution of myosin rod protein to the structural organization of adult and embryonic muscles in Drosophila

Myosin rod protein (MRP) is a naturally occurring 155 kDa protein in Drosophila that includes the myosin heavy chain (MHC) rod domain, but contains a unique 77 amino acid residue N-terminal region that replaces the motor and light chain-binding domains of S1. MRP is a major component of myofilaments in certain direct flight muscles (DFMs) and it is present in other somatic, cardiac and visceral muscles in adults, larvae and embryos, where it is coexpressed and polymerized into thick filaments along with MHC. DFM49 has a relatively high content of MRP, and is characterized by an unusually disordered myofibrillar ultrastructure, which has been attributed to lack of cross-bridges in the filament regions containing MRP. Here, we characterize in detail the structural organization of myofibrils in adult and embryonic Drosophila muscles containing various MRP/MHC ratios and in embryos carrying a null mutation for the single MHC gene. We examined MRP in embryonic body wall and intestinal muscles as well as in DFMs with consistent findings. In DFMs numbers 49, 53 and 55, MRP is expressed at a high level relative to MHC and is associated with disorder in the positioning of thin filaments relative to thick filaments in the areas of overlap. Embryos that express MRP in the absence of MHC form thick filaments that participate in the assembly of sarcomeres, suggesting that myofibrillogenesis does not depend on strong myosin-actin interactions. Further, although thick filaments are not well ordered, the relative positioning of thin filaments is fairly regular in MRP-only containing sarcomeres, confirming the hypothesis that the observed disorder in MRP/MHC containing wild-type muscles is due to the combined action between the functional behavior of MRP and MHC myosin heads. Our findings support the conclusion that MRP has an active function to modulate the contractile activity of muscles in which it is expressed.     

Relation of nebulin and connectin (titin) to dynamics of actin in nascent myofibrils of cultured skeletal muscle cells.

Cultured embryonic chicken skeletal muscle cells microinjected with rhodamine (rh)-labeled actin were stained with antibodies against nebulin and connectin (titin). In premyofibril areas, nebulin was observed as dotted structures, many of which were arranged in a linear fashion. These structures were associated with injected rh-actin. Among these linearly arranged dots of nebulin and rh-actin, numerous small nebulin dots without rh-actin incorporation were scattered. It is probable that the dots of nebulin and/or its associated protein(s) represent a preformed scaffold upon which actin monomers accumulate; exogenously introduced actin associates initially with small nebulin dots, which in turn coalesce to form rh-actin dots and are arranged linearly. In developing myofibrils, two patterns of nebulin distribution were found: "singlets" and "doublets." Recovery of rh-actin's fluorescence after photobleaching was slowest in the nonstriated dotted portions, followed by the striated myofibrillar portions with nebulin singlets and those with doublets, in that order. Thus, the distribution patterns of nebulin seem to be related to the accessibility/exchangeability of actin into nascent myofibrils. It is possible that early nebulin filaments exhibiting singlets are not tightly associated with actin filaments and that this loose association allows myofibrils to exchange nonadult isoforms of actin and other proteins into adult types. Connectin formed a striated pattern before the formation of rh-actin/nebulin striations. It appears that connectin does not have any significant role in the accessibility of actin into nascent myofibrils.     

Mef2s are required for thick filament formation in nascent muscle fibres

During skeletal muscle differentiation, the actomyosin motor is assembled into myofibrils, multiprotein machines that generate and transmit force to cell ends. How expression of muscle proteins is coordinated to build the myofibril is unknown. Here we show that zebrafish Mef2d and Mef2c proteins are required redundantly for assembly of myosin-containing thick filaments in nascent muscle fibres, but not for the earlier steps of skeletal muscle fibre differentiation, elongation, fusion or thin filament gene expression. mef2d mRNA and protein is present in myoblasts, whereas mef2c expression commences in muscle fibres. Knockdown of both Mef2s with antisense morpholino oligonucleotides or in mutant fish blocks muscle function and prevents sarcomere assembly. Cell transplantation and heat-shock-driven rescue reveal a cell-autonomous requirement for Mef2 within fibres. In nascent fibres, Mef2 drives expression of genes encoding thick, but not thin, filament proteins. Among genes analysed, myosin heavy and light chains and myosin-binding protein C require Mef2 for normal expression, whereas actin, tropomyosin and troponin do not. Our findings show that Mef2 controls skeletal muscle formation after terminal differentiation and define a new maturation step in vertebrate skeletal muscle development at which thick filament gene expression is controlled.     

Easily releasable myofilaments from skeletal and cardiac muscles maintained in vitro. Role in myofibrillar assembly and turnover

Gentle treatment with an ATP-containing relaxing solution of isolated myofibrils from rat diaphragm, soleus, extensor digitorum longus, and left atria maintained in vitro releases a small amount of myofilaments constituting less than 5% of total myofibrillar protein. Successive extraction of myofibrils produced little further filament release. Releasable myofilaments lack alpha-actinin (Mr = 95,000), certain very high molecular weight proteins (greater than 200,000), and possibly M-line protein but contain other myofibrillar proteins. After pulse-labeling with [3H]leucine for 8 min, specific activity of the myosin heavy chain in the easily releasable myofilaments is 3-6 times higher than the specific activity of myosin heavy chain in the residual myofibrils, although 85-90% of total label is in the myofibrillar myosin. In the absence of protein synthesis, releasable filament specific activity decreases, with a half-time of 60-90 min, to that of the myofibrillar myosin. This labeling pattern appears inconsistent with a simple precursor-product relationship between releasable filaments and myofibrils suggesting that the filaments originate largely from myofibrils. Preincubation of muscles with several factors known to decrease proteolysis, i.e. passive stretch, leupeptin, colchicine, and cycloheximide, reduced the size of the releasable filament fraction. Treatment of muscles with the calcium ionophore A23187, which accelerates proteolysis, and pretreatment of myofibrils with either trypsin or calcium-dependent protease increased filament release. Therefore, the releasable filament fraction may contain intermediates in the breakdown of myofibrils. The labeling kinetics may indicate a mixing of myofilaments within myofibrils which functions in the movement of contractile protein to its possible site of degradation, i.e. the myofibrillar surface.     

Purified thick filaments from the nematode Caenorhabditis elegans: evidence for multiple proteins associated with core structures

The thick filaments of the nematode, Caenorhabditis elegans, arising predominantly from the body-wall muscles, contain two myosin isoforms and paramyosin as their major proteins. The two myosins are located in distinct regions of the surfaces, while paramyosin is located within the backbones of the filaments. Tubular structures constitute the cores of the polar regions, and electron-dense material is present in the cores of the central regions (Epstein, H.F., D.M. Miller, I. Ortiz, and G.C. Berliner. 1985. J. Cell Biol. 100:904-915). Biochemical, genetic, and immunological experiments indicate that the two myosins and paramyosin are not necessary core components (Epstein, H.F., I. Ortiz, and L.A. Traeger Mackinnon. 1986. J. Cell Biol. 103:985-993). The existence of the core structures suggests, therefore, that additional proteins may be associated with thick filaments in C. elegans. To biochemically detect minor associated proteins, a new procedure for the isolation of thick filaments of high purity and structural preservation has been developed. The final step, glycerol gradient centrifugation, yielded fractions that are contaminated by, at most, 1-2% with actin, tropomyosin, or ribosome-associated proteins on the basis of Coomassie Blue staining and electron microscopy. Silver staining and radioautography of gel electrophoretograms of unlabeled and 35S-labeled proteins, respectively, revealed at least 10 additional bands that cosedimented with thick filaments in glycerol gradients. Core structures prepared from wild-type thick filaments contained at least six of these thick filament-associated protein bands. The six proteins also cosedimented with thick filaments purified by gradient centrifugation from CB190 mutants lacking myosin heavy chain B and from CB1214 mutants lacking paramyosin. For these reasons, we propose that the six associated proteins are potential candidates for putative components of core structures in the thick filaments of body-wall muscles of C. elegans.     

Assembly of thick filaments and myofibrils occurs in the absence of the myosin head

We investigated the importance of the myosin head in thick filament formation and myofibrillogenesis by generating transgenic Drosophila lines expressing either an embryonic or an adult isoform of the myosin rod in their indirect flight muscles. The headless myosin molecules retain the regulatory light-chain binding site, the alpha-helical rod and the C-terminal tailpiece. Both isoforms of headless myosin co-assemble with endogenous full-length myosin in wild-type muscle cells. However, rod polypeptides interfere with muscle function and cause a flightless phenotype. Electron microscopy demonstrates that this results from an antimorphic effect upon myofibril assembly. Thick filaments assemble when the myosin rod is expressed in mutant indirect flight muscles where no full-length myosin heavy chain is produced. These filaments show the characteristic hollow cross-section observed in wild type. The headless thick filaments can assemble with thin filaments into hexagonally packed arrays resembling normal myofibrils. However, thick filament length as well as sarcomere length and myofibril shape are abnormal. Therefore, thick filament assembly and many aspects of myofibrillogenesis are independent of the myosin head and these processes are regulated by the myosin rod and tailpiece. However, interaction of the myosin head with other myofibrillar components is necessary for defining filament length and myofibril dimensions.